Although there is compelling evidence for the presence of bipotent progenitor cells in the adult liver, the role of these progenitors s in hepatocarcinogenesis is still a subject of debate. Building on our past experience with cholangiocytes and oval cells, bipotent biliary progenitors activated by most hepatocarcinogens, we have continued with our efforts to identify and characterize bipotent progenitors present in the biliary tree and to ascertain their role in hepato-and cholangio-carcinogenesis. Over the past 4 years, our studies of cholangiocyte marker positive (CMP), bipotent, fetal liver epithelial cells (FLEC) have yielded novel monoclonal antibody based schemes for isolating bipotent CMP-FLEC. Results from transplantation of CMP-FLEC isolates from dipeptidyl peptidase IV (DPPIV) positive rats into DPPIV deficient host rats treated with retrorsine/partial hepatectomy (R/PH) led to the unexpected finding that CMP-FLEC possesses a much higher capacity for growth in the R/PH treated adult liver than fetal hepatoblasts and other nonparenchymal cell types. In the current proposal, isolation schemes developed for bipotent CMP-FLEC will be applied to the isolation of phenotypically equivalent CMP-liver epithelial cells (CMP-LEC) from newborn and adult rat liver. We hypothesize that these fetal-like CMP-FLEC will possess a capacity for hepatocytic differentiation similar to oval cells and will retain this capacity following spontaneous transformation in vitro and progression to HCC in vivo. In Specific Aim 1, we will employ a rapid transplantation model that replaces retrorsine with mitomycin C (mitoC/PH) to test the hypothesis that the expression of the cholangiocyte marker OC.4, a marker first seen at 2 after birth, identifies mature CMP-LEC that have a greatly diminished capacity for hepatocytic differentiation. In Specific Aim 2, we will test the hypothesis that spontaneously transformed CMP-LEC and oval cells but not BDEC will undergo incomplete hepatocytic differentiation in mitoC/PH treated rats and progress to CMP-HCC. Spontaneous transformation of CMP-LEC will be accelerated by selection on plastic and/or soft agar, transfection with ErbB2 or exposure to carcinogen in situ. In Specific Aim 3, genomic and proteomic methodologies will be used to map the signaling pathways operative during the spontaneous transformation of BDEC, CMP-LEC and oval cells that promote survival and cooperate with ErbB2 to confer anchorage independent/invasive growth. Specific Aim 4 will continue with the characterization of BD.1, a 170 kDa protein expressed by cholangiocytes but not oval cells that forms stable complexes with CLIP170, a microtubule associated protein. We will test the hypothesis that loss of BD.1 alters microtubule/kinetochore dynamics in a manner that promotes aneuploidy. The proposed studies will provide new insights into the role of bipotent progenitors in liver cancer.